2.5. ELISA Analysis

Blood samples were centrifuged at 2000 rpm for 20 min, and serum was collected and stored at −20 °C until processing. Quantikine® ELISA kits from R&D systems and Biotechne were used. Each kit was specific for vascular endothelial growth factor (VEGF, Catalog no. MMV00), VEGFR1 or Flt-1 (Catalog no. MVR100), and placental growth factor (PlGF2, Catalog no. MP200). According to the manufacturer’s information, the mouse VEGFR1/Flt-1 ELISA kit measures natural and recombinant mouse sVEGFR1 (sFlt-1). Maternal serum was diluted with the dilution buffer included in the kit as follows: 1:20 for Flt-1, 1:4 for VEGF, and 1:4 for PlGF. Standards and samples were assayed in duplicate in 96-well plates coated with the appropriate polyclonal antibody following the manufacturer’s instructions.